Challenges and stepwise fit-for-purpose optimization for bioanalyses of remdesivir metabolites nucleotide monophosphate and triphosphate in mouse tissues using LC–MS/MS

[Display omitted] •A robust and reliable LC–MS/MS method for the quantification of RMP and RTP was optimized and validated.•The novel method solved the major challenges of direct determination of RMP and RTP in biological matrix through improvement of LC retention, stability and recovery.•The method was validated and successfully applied to mouse tissue distribution study.•This method provides useful information for further study of remdesivir as well as extends the approach for phosphate determination.•The method solved the major challenges for determining RMP and RTP in biological matrix such as LC retention, stability and recovery. Remdesivir is a prodrug of the nucleotide analogue and used for COVID-19 treatment. However, the bioanalysis of the active metabolites remdesivir nucleotide triphosphate (RTP) and its precursor remdesivir nucleotide monophosphate (RMP) is very challenging. Herein, we established a novel method to separate RTP and RMP on a BioBasic AX column and quantified them by high-performance liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. Stepwise, we optimized chromatographic retention on an anion exchange column, improved stability in matrix through the addition of 5,5′-dithiobis-(2nitrobenzoic acid) and PhosSTOP EASYpack, and increased recovery by dissociation of tight protein binding with 2 % formic acid aqueous solution. The method allowed lower limit of quantification of 20 nM for RMP and 10 nM for RTP. Method validation demonstrated acceptable accuracy (93.6%–103% for RMP, 94.5%–107% for RTP) and precision (RSD < 11.9 % for RMP, RSD < 11.4 % for RTP), suggesting that it was sensitive and robust for simultaneous quantification of RMP and RTP. The method was successfully applied to analyze RMP and RTP in mouse tissues. In general, the developed method is suitable to monitor RMP and RTP, and provides a useful approach for exploring more detailed effects of remdesivir in treating diseases.